Inhibition of Biofilm Organisms

ABSTRACT

The present invention relates to a product comprising at least two antibiofilm agents wherein at least one of the antibiofilm agents is an antimicrobial peptide. The second antibiofilm agent is cysteamine. There is also provided the use of the product in the treatment of a microbial infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present invention is a continuation of U.S. application Ser. No.13/260,547 filed Nov. 17, 2011 which is a U.S. National Phaseapplication of PCT Patent Application No. PCT/GB2010/000631, filed onMar. 31, 2010 and claims priority to United Kingdom Patent ApplicationNo. GB 0905451.1 filed on Mar. 31, 2009, which claims priority to U.S.Provisional Patent Application No. 61/165,396, filed on Mar. 31, 2009,the disclosures of which are incorporated by reference in their entiretyfor all purposes.

FIELD OF THE INVENTION

The invention relates to products, compositions, methods and uses whichare useful in relation to the prevention and treatment of biofilms.

BACKGROUND OF THE INVENTION

A microbial biofilm is a community of microbial cells embedded in anextracellular matrix of polymeric substances and adherent to abiological or a non-biotic surface. A range of microorganisms (bacteria,fungi, and/or protozoa, with associated bacteriophages and otherviruses) can be found in these biofilms. Biofilms are ubiquitous innature, are commonly found in a wide range of environments. Biofilms arebeing increasingly recognised by the scientific and medical community asbeing implicated in many infections, and especially their contributionto the recalcitrance of infection treatment.

Biofilms are etiologic agents for a number of disease states in mammalsand are involved in 80% of infections in humans. Examples include skinand wound infections, middle-ear infections, gastrointestinal tractinfections, peritoneal membrane infections, urogenital tract infections,oral soft tissue infections, formation of dental plaque, eye infections(including contact lens contamination), endocarditis, infections incystic fibrosis, and infections of indwelling medical devices such asjoint prostheses, dental implants, catheters and cardiac implants.

Planktonic microbes (i.e., microorganisms suspended or growing in aliquid medium) are typically used as models for antimicrobialsusceptibility research as described by the Clinical and LaboratoryStandards Institute (CLSI) and European Committee on AntimicrobialSusceptibility Testing (EUCAST). Microbes in biofilms are significantlymore resistant to antimicrobial treatment than their planktoniccounterparts. However, there is no standardized method for the study ofantibiotic susceptibility of biofilm microbes.

Biofilm formation is not limited solely to the ability of microbes toattach to a surface. Microbes growing in a biofilm are able to interactmore between each other than with the actual physical substratum onwhich the biofilm initially developed. For example, this phenomenonfavours conjugative gene transfer, which occurs at a greater ratebetween cells in biofilms than between planktonic cells. This representsan increased opportunity for horizontal gene transfer between bacteria,and is important because this can facilitate the transfer of antibioticresistance or virulence determinant genes from resistant to susceptiblemicrobes. Bacteria can communicate with one another by a system known asquorum sensing, through which signalling molecules are released into theenvironment and their concentration can be detected by the surroundingmicrobes. Quorum sensing enables bacteria to co-ordinate theirbehaviour, thus enhancing their ability to survive. Responses to quorumsensing include adaptation to availability of nutrients, defense againstother microorganisms which may compete for the same nutrients and theavoidance of toxic compounds potentially dangerous for the bacteria. Itis very important for pathogenic bacteria during infection of a host(e.g. humans, other animals or plants) to co-ordinate their virulence inorder to escape the immune response of the host in order to be able toestablish a successful infection.

Biofilm formation plays a key role in many infectious diseases, such ascystic fibrosis and periodontitis, in bloodstream and urinary tractinfections and as a consequence of the presence of indwelling medicaldevices. The suggested mechanisms by which biofilm-associatedmicroorganisms elicit diseases in their host include the following: (i)delayed penetration of the antimicrobial agent through the biofilmmatrix, (ii) detachment of cells or cell aggregates from indwellingmedical device biofilms, (iii) production of endotoxins, (iv) resistanceto the host immune system, (v) provision of a niche for the generationof resistant organisms through horizontal gene transfer of antimicrobialresistance &/or virulence determinant genes, and (vi) altered growthrate (i.e. metabolic dormancy) (Donlan and Costerton, Clin Microbiol Rev15: 167-193, 2002; Parsek and Singh, Annu Rev Microbiol 57: 677-701,2003; Costerton J W, Resistance of biofilms to stress. In ‘The biofilmprimer’. (Springer Berlin Heidelberg). pp. 56-64.2007).

Recent experimental evidence has indicated the existence within biofilmsof a small sub-population of specialized non-metabolising persistercells (dormant cells). It is thought that these cells may be responsiblefor the high resistance/tolerance of biofilm to antimicrobial agents.Multi-drug-tolerant persister cells are present in both planktonic andbiofilm populations and it appears that yeasts and bacteria have evolvedanalogous strategies that assign the function of survival to thissub-population. The protection offered by the polymeric matrix allowspersister cells to evade elimination and serve as a source forre-population. There is evidence that persisters may be largelyresponsible for the multi-drug tolerance of microbial biofilms (LaFleuret al., Antimicrob Agents Chemother. 50: 3839-46, 2006; Lewis, NatureReviews Microbiology 5, 48-56 2007).

There remains a need for better therapies for preventing biofilmformation and treating conditions associated with microbial biofilms.

DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of Examples only withreference to the following Figures in which:

FIG. 1: Antibacterial activity of NP108 and NM001 (cysteamine) againstP. aeruginosa ATCC BAA-47 planktonic cells

FIG. 2: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against P. aeruginosa ATCC BAA-47 planktonic cells

FIG. 3: Antibacterial activity of NP108 and NM001 (cysteamine) againstS. aureus DSM 11729 planktonic cells

FIG. 4: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against S. aureus DSM 11729 planktonic cells

FIG. 5: Activity of NP339 against biofilm cells of Gram-positive andGram-negative bacteria

FIG. 6: Activity of NP339 against persister cells of Gram-positive andGram-negative bacteria

FIG. 7: Activity of NP341 against biofilm cells of Gram-positive andGram-negative bacteria

FIG. 8: Activity of NP341 against persister cells of Gram-positive andGram-negative bacteria

FIG. 9: Activity of NM001 (cysteamine) against biofilm cells ofGram-positive and Gram-negative bacteria

FIG. 10: Activity of NM001 (cysteamine) against persister cells ofGram-positive and Gram-negative bacteria

FIG. 11: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against P. aeruginosa ATCC BAA-47 biofilm cells

FIG. 12: Antibacterial activity of NP108 and NM001 (cysteamine) againstP. aeruginosa ATCC BAA-47 persister cells

FIG. 13: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against P. aeruginosa ATCC BAA-47 persister cells

FIG. 14A-14D: Antibacterial activity of NP339 and NM001 (cysteamine)combinations against (a) P. aeruginosa DSM 1128, (b) P. aeruginosa ATCCBAA-47, (c) P. aeruginosa DSM 1299 and (d) P. aeruginosa ATCC 27853biofilm cells

FIG. 15A-15B: Antibacterial activity of NP339 and NM001 (cysteamine)combinations against (a) P. aeruginosa DSM 1128 and (b) P. aeruginosaATCC BAA-47 persister cells

FIG. 16: Antibacterial activity of NP108 and NM001 (cysteamine) againstS. aureus DSM 11729 biofilm cells

FIG. 17: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against S. aureus DSM 11729 biofilm cells

FIG. 18: Antibacterial activity of NP108 and NM001 (cysteamine) againstS. aureus DSM 11729 persister cells

FIG. 19: Antibacterial activity of NP108 and NM001 (cysteamine)combinations against S. aureus DSM 11729 persister cells

FIGS. 20A-20B and 21A-21B: Activity of the mucolytic agentsN-acetylcysteine (FIGS. 20(a) and 20(b)) and NM001 (cysteamine) (FIGS.21(a) and 21(b)) alone and in combination with NP341 against P.aeruginosa 27853 planktonic cells

FIG. 22A: untreated control S. aureus biofilm after 24 hours

FIG. 22B: S. aureus biofilm 24 hours following treatment with NM001(cysteamine) at 2 mg/ml

FIG. 22C: S. aureus biofilm 24 hours following treatment with Colistinat 0.2 mg/ml

FIG. 22D: S. aureus biofilm 24 hours following treatment with peptideNP108 at 2 mg/ml

FIG. 23A: untreated control S. aureus biofilm after 24 hours

FIG. 23B: S. aureus biofilm 24 hours following treatment with NM001(cysteamine) at 2 mg/ml

FIG. 23C: S. aureus biofilm 24 hours following treatment with Colistinat 0.2 mg/ml

FIG. 23D: S. aureus biofilm 24 hours following treatment with peptideNP108 at 2 mg/ml

FIG. 24A: untreated control P. aeruginosa biofilm after 24 hours

FIG. 24B: P. aeruginosa biofilm 24 hours following treatment with NM001(cysteamine) at 2 mg/ml

FIG. 24C: P. aeruginosa biofilm 24 hours following treatment withColistin at 0.2 mg/ml

FIG. 24D: P. aeruginosa biofilm 24 hours following treatment withpeptide NP108 at 2 mg/ml

FIG. 25A: untreated control P. aeruginosa biofilm after 24 hours

FIG. 25B: P. aeruginosa biofilm 24 hours following treatment with NM001(cysteamine) at 2 mg/ml

FIG. 25C: P. aeruginosa biofilm 24 hours following treatment withColistin at 0.2 mg/ml

FIG. 25D: P. aeruginosa biofilm 24 hours following treatment withpeptide NP108 at 2 mg/ml

FIG. 26: Activity of NP432 alone and in combination with NM001(cysteamine) or in combination with NP108 against P. aeruginosa PAO1biofilm

FIG. 27: Activity of NP445 alone and in combination with NM001(cysteamine) or in combination with NP108 against P. aeruginosa PAO1biofilm

FIG. 28: Activity of NP458 alone and in combination with NM001(cysteamine) or in combination with NP108 against P. aeruginosa PAO1biofilm

FIG. 29: Activity of NP462 alone and in combination with NM001(cysteamine) or in combination with NP108 against P. aeruginosa PAO1biofilm

FIG. 30: Activity of NP432 alone and in combination with NM001(cysteamine) or in combination with NP108 against S. aureus ATCC25923biofilm

FIG. 31: Activity of NP445 alone and in combination with NM001(cysteamine) or in combination with NP108 against S. aureus ATCC25923biofilm

FIG. 32: Activity of NP458 alone and in combination with NM001(cysteamine) or in combination with NP108 against S. aureus ATCC25923biofilm

FIG. 33: Activity of NP462 alone and in combination with NM001(cysteamine) or in combination with NP108 against S. aureus 25923biofilm

Table 1: Summary of the activity of the tested antimicrobial agentsagainst the Gram-negative P. aeruginosa strains and the Gram-positiveStaphylococcus spp.

Table 2: Summary of the activity of the tested antimicrobial agentsagainst S. epidermidis, S. aureus, and P. aeruginosa.

The present invention relates to a product comprising at least twoantibiofilm agents wherein at least one of the antibiofilm agents is anantimicrobial peptide. The second antibiofilm agent is generally adispersant or an anti-adhesive agent. There is also provided the use ofthe product in the treatment of a microbial infection.

TABLE 1 Exp#1-2 MIC (μg/ml) at pH 7 S. S. S. P. P. P. epidermidis aureusaureus aeruginosa aeruginosa aeruginosa NP Sequence ATCC12228 ATCC25923DSMZ11729 DSMZ1128 DSMZ1299 ATCCBAA-47 NP432 RRRFRFFFRFRRR <7.8 31.2562.5 62.5 15.6 15.6 NP438 HHHFRFFFRFRRR <7.8 >500 >500 >500 500 >500NP441 HHPRRKPRRPKRHH >500 >500 >500 >500 >500 >500 NP445 KKFPWRLRLRYGRR<7.8 500 500 62.5 31.25 31.25 NP449 KKPRRKPRRPKRKK- 31.25 250 125 250125 250 cyst NP451 HHPRRKPRRPKRHH- 125 500 500 >500 >500 >500 cyst NP457RRRRR-cyst 31.25 125 125 >500 >500 250 NP458 RRRRRHH-cyst 31.25 250 250250 125 62.5

TABLE 2 MBC (μg/ml) following MIC at pH 7 Exp#3 P. aeruginosa S.epidermidis S. aureus S. aureus P. aeruginosa P. aeruginosa P.aeruginosa P. aeruginosa NP DSMZ1299 ATCC12228 ATCC25923 DSMZ11729DSMZ1128 DSMZ1299 ATCCBAA-47 DSMZ1299 NP432 16 250 500 250 (2)  32 (2)250 NP438 125 >500 >500 >500 >500 >500NP441 >500 >500 >500 >500 >500 >500 >500 >500 NP445 62.5 >500 >500  250 125 250 NP449 125 >500 250 500 (2) 250 (2) >500 NP451 >500125 >500 >500 >500 >500 >500 >500 NP457 >500 125 (2) 62.5 125(2) >500 >500 >500 >500 NP458 500 125 250 >500 >500 >500 Exp#3 Exp#4 MIC(μg/ml) pH 5.5, Exp#4 MBC (μg/ml) pH 5.5, MIC (μg/ml) pH 5.5 320 mM NaClMBC (μg/ml) at pH 5.5 320 mM NaCl S. aureus P. aeruginosa S. aureus P.aeruginosa S. aureus P. aeruginosa S. aureus P. aeruginosa NP 11729ATCCBAA-47 11729 ATCCBAA-47 11729 ATCCBAA-47 11729 ATCCBAA-47 NP432 >500125 >500 125 >500 125 >500 >500 NP438 >500 125 >50062.5 >500 >500 >500 >500 NP441 >500 >500 >500 >500 >500 >500 >500 >500NP445 >500 >500 >500 >500 >500 >500 >500 >500NP449 >500 >500 >500 >500 >500 >500 >500 >500NP451 >500 >500 >500 >500 >500 250 >500 >500NP457 >500 >500 >500 >500 >500 >500 >500 >500NP458 >500 >500 >500 >500 >500 >500 >500 >500

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is provided aproduct comprising at least two antibiofilm agents wherein at least oneof the antibiofilm agents is an antimicrobial peptide. The otherantibiofilm agent may be a dispersant or an anti-adhesive agent.

The term “antibiofilm agent” is used herein to describe an agent that iscapable of destroying or inhibiting the growth of a microbial biofilm.The antibiofilm agent may be capable of disrupting the structure of thebiofilm, for example the extracellular mucous matrix, or may be capableof destroying or inhibiting the growth of microbial cells within thebiofilm.

The invention further provides a method of preventing biofilm formationin an environment comprising the step of administering to theenvironment an antimicrobial peptide. Advantageously the methodcomprises the step of administering to the environment a productaccording to the invention.

The invention further provides a method for treating a microbialinfection, particularly a microbial biofilm, by prophylaxis or therapy,comprising the administration in a therapeutically effective amount ofan antimicrobial peptide, for example a cationic peptide. Typically themethod involves the sequential or combined administration in atherapeutically effective amount of:

a first antibiofilm agent; and

a second antibiofilm agent different from the first one; wherein atleast one of the first and second antibiofilm agents is an antimicrobialpeptide for example a cationic peptide.

The above mentioned active agents may be administered as free or fixedcombinations. Free combinations may be provided as combination packagescontaining all the active agents in free combinations. Fixedcombinations are often tablets or capsules.

Included in the invention is the use in the manufacture of a medicamentfor the treatment of a microbial infection, particularly a microbialbiofilm infection, by prophylaxis or therapy of the antimicrobialpeptides, or combinations of active agents outlined above.

The products have the advantage that they demonstrate antibacterialactivity against, inter alia, the persister cells present in thebiofilms, which is an essential step towards the eradication ofbiofilms.

The agents of the invention may be administered in the form ofpharmaceutically acceptable salts. The pharmaceutically acceptable saltsof the present invention can be synthesized from the parent compoundwhich contains a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting the free acidor base forms of these compounds with a stoichiometric amount of theappropriate base or acid in water or in an organic solvent, or in amixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, thedisclosure of which is hereby incorporated by reference; see also Stahlet al, Eds, “Handbook of Pharmaceutical Salts Properties Selection andUse”, Verlag Helvetica Chimica Acta and Wiley-VCH, 2002. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or, as the case may be, an animalwithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The invention thus includes pharmaceutically-acceptable salts of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof for example the conventional non-toxic saltsor the quaternary ammonium salts which are formed, e.g., from inorganicor organic acids or bases. Examples of such acid addition salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base saltsinclude ammonium salts, alkali metal salts such as sodium and potassiumsalts, alkaline earth metal salts such as calcium and magnesium salts,salts with organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, and so forth. Also, the basic nitrogen-containing groups may bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethyl bromidesand others.

The invention therefore includes pharmaceutical products generallycomprising at least:

a first antibiofilm agent; and

a second antibiofilm agent different from the first one wherein at leastone of the first and second antibiofilm agents is an antimicrobialpeptide for example a cationic peptide.

The First Antibiofilm Agent

The first antibiofilm agent may be an antimicrobial peptide for examplean antibacterial peptide. Preferably the first antibiofilm agent is anantimicrobial peptide, hereinafter referred to as “the firstantimicrobial agent”. The first antimicrobial agent may comprise aminoacids according to the formula I:

((X)_(l)(Y)_(m))_(n)  (I)

wherein l and m are integers from 1 to 10, for example 1 to 5; n is aninteger from 1 to 10; X and Y, which may be the same or different, areindependently a hydrophobic or cationic amino acid.

Preferably the first antimicrobial agent comprises amino acids accordingto the formula (I) wherein X and Y are cationic amino acids.

The antimicrobial peptide may comprise from 2 to 200 amino acids, forexample 3, 4, 5, 6, or 7 up to 100 amino acids, including 3, 4, 5, 6, or7 up to 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids. According toone embodiment, the antimicrobial peptide comprises 3 or 4 to 50 aminoacids. Alternatively the peptide may comprise more than 27 amino acids,typically 27 to 300 amino acids, suitably 27 to 200 amino acids.

The peptide may comprise 100 to 200 amino acids, 20 to 100, 20 and 45amino acids such as 20, 25, 30, 35, 40, 42 or 45 amino acids. Thepeptide may comprise between 3 and 15 amino acids, for example 5 to 15amino acids.

As used herein, the term “hydrophobic” refers to an amino acid having aside chain that is uncharged at physiological pH, that is not polar andthat is generally repelled by aqueous solution.

As used herein, the term “cationic” refers to amino acids having a netcharge that is greater than or equal to 0. Generally the term “cationic”refers to amino acids having a net charge that is greater than zero.

Generally a hydrophobic amino residue has a hydrophobicity greater thanor equal to −1.10 and a charge greater than or equal to 0.

Hydrophobic amino acids may include, leucine phenylalanine, proline,alanine, tryptophan, valine, isoleucine and methionine.

Preferably X and/or Y are cationic amino acids for example selected fromthe group consisting of histidine, arginine and lysine. Preferably stillX and/or Y are arginine or lysine. X and/or Y may be selected fromnon-naturally occurring amino acids for example the cationic amino acidornithine.

X and/or Y may be optical isomers of a cationic amino acid as definedherein for example D or L-amino acids. Moreover, X and/or Y may bealternating amino acids.

The amino acids may be naturally occurring or synthetic. The inventionalso includes known isomers (structural, stereo-, conformational &configurational) and structural analogues of the above amino acids, andthose modified either naturally (e.g. post-translational modification)or chemically, including, but not exclusively, phosphorylation,glycosylation, sulfonylation and/or hydroxylation.

According to one embodiment the peptide may include one or moresubstitution of the cationic or hydrophobic amino acids X and Y.However, the peptide would predominantly comprise the cationic orhydrophobic amino acids X and Y. Typically the peptide may comprise 1 to5 substitutions, suitably 1 to 3 substitutions, generally onesubstitution. The substitutions may be terminal or non-terminal.

The substitutions may consist of amino acids, or non-amino acids. Thesubstitutions may be charged or uncharged. Typically one or more of thesubstitutions are uncharged amino acids. Alternatively or additionallyone or more of the substitutions may be non-amino acids such ascysteamine.

Preferably X and Y are the same and are lysine or arginine.

According to one embodiment, the peptide comprises predominantlyarginine amino acids which may be substituted with one or more aminoacids which are not arginine.

Generally the peptide comprises 7 to 20 arginine amino acids, optionallysubstituted with 1 to 5 non-arginine amino acids, typically 3 to 5non-arginine substitutions.

Alternatively the peptide may comprise 7 to 20 lysine amino acids,optionally substituted with 1 to 5 non-lysine amino acids, typically 3to 5 non-lysine substitutions.

According to a further embodiment, the peptide may comprise 27 to 300lysine amino acids, generally 27 to 200 lysine amino acids. Typicallythe peptide comprises no non-terminal substitutions with non-lysineamino acids.

In the peptide of formula (I) l and m may be 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In the peptide of formula (I) l may be 1, n may be 1 and m may bebetween 4 and 9, for example, m may be 3, 4, 5, 6, 7, 8 or 9.

In the peptide of formula (I) l, n and/or m may be between 1 and 5, forexample, 1, 2, 3, 4 or 5.

In the peptide of formula (I) l and m may be an integer between 0 and 7and n may be an integer between 1 and 10.

In the peptide of formula (I) l and m may be 0, 1 or 2 and n may be aninteger between 1 and 10.

In the peptide of formula (I) X and Y may be the same, l may be 0, m maybe 1 and n may be 3, 4, 5, 6, 7, 8, 9 or 10.

In the peptide of formula (I) X and Y may be the same, l and m may be 1and n may be 2, 3, 4 or 5.

In the peptide of formula (I) X and Y may be the same, l may be 1, m maybe 2 and n may be 1, 2, 3 or 4.

In the peptide of formula (I) X and Y may be the same, l and m may be 2and n may be 1, 2, 3 or 4.

Preferably the first antimicrobial agent comprises a peptide sequenceselected from the group consisting of polylysine and polyarginine.

In one embodiment, the first antimicrobial agent comprises a polylysine.

In an alternative embodiment, the first antimicrobial agent comprisespolyarginine.

According to a further aspect of the present invention there is providedthe use of the first antimicrobial agent in the treatment of preventionof a biofilm.

Typically the first antimicrobial agent is in the form of the product ofthe invention as described below.

The Second Antibiofilm Agent

The second antibiofilm agent may be any agent which inhibits biofilmformation. By way of example, the second antibiofilm agent may inhibitbacterial adhesion, hydrophobicity or slime production. The secondantibiofilm agent may be selected from a dispersant and an anti-adhesiveagent.

According to one embodiment of the present invention the secondantibiofilm agent is not a peptide.

The term “dispersant” is intended to include any agent capable ofdispersing the particles of a biofilm. In particular, the dispersant maypromote the dispersion of slime produced by microbes such as bacteria,mucous which forms part of the biofilm for example mucous produced bythe cells to which the biofilm microbes adheres, and biofilm microbessuch as bacteria.

The dispersant may be a mucolytic agent. The mucolytic agent may be anenzyme for example a DNase, alginase, protease or carobohydrase.Alternatively the mucolytic agent may be a small molecule for example anamine such as an aminothiol or an acid such asethylenediaminetetraacetic acid (EDTA). The amine may be selected fromacetylcysteine and cysteamine.

The term “anti-adhesive agent” is intended to include any agent capableof inhibiting adhesion between cells, proteins and organisms e.g.microbes thereby preventing biofilm formation or promoting biofilmself-destruction. In particular, the anti-adhesive agent may prevent theadhesion to a surface or substrate of all cell types encountered inmicrobial biofilms in particular free living microbes i.e. planktoniccells. Anti-adhesive agents may include, but are not limited to,hyaluronan, heparin or Carbopol 934.

The second antibiofilm agent may be an antibacterial agent. Theantibacterial agent may be a mucolytic agent for example a mucolyticagent having both mucolytic and antibacterial activity. Preferably theantibacterial agent is cysteamine.

The Products of the Invention

The product of the present invention may comprise an antimicrobialpeptide.

A preferred product comprises an antimicrobial peptide and a mucolyticagent.

The ratio of the first antibiofilm agent to the second antibiofilm agentin the products of the invention may be from 1:10 to 10:1; generally atleast 2:1 for example at least 3:1 or 4:1. According to one embodiment,the ratio of first antibiofilm agent to the second antibiofilm agent isapproximately 1:1. Preferably the first antibiofilm agent is a cationicpeptide and the second antibiofilm agent is a mucolytic agent and theratio of cationic peptide:mucolytic agent ranges from 2:1 up to 4:1.According to a further embodiment the ratio may be approximately 1:1.

The active agents may be administered simultaneously, sequentially orseparately. The active agents may be provided as a combination package.The combination package may contain the product of the inventiontogether with instructions for simultaneous, separate or sequentialadministration of each of the active agents. For sequentialadministration, the active agents can be administered in any order.

The active agents of the product of the invention may be provided aspharmaceutical compositions additionally containing one or morepharmaceutically acceptable diluents, excipients and/or carriers. Thisapplies to both fixed and free combinations.

The active agents of the present invention may be administered by anysuitable route known to those skilled in the art, preferably in the formof a pharmaceutical composition adapted to such a route, and in a doseeffective for the treatment intended. The active compounds andcomposition may, for example, be administered parenterally, orally,intranasal, intrabronchial, enterally, transdermally, sublingually,rectally, vaginally, ocularly, or topically. Both local and systemicadministration is contemplated.

For the purposes of parenteral administration (“parenteral” as usedherein, refers to modes of administration which include intravenous,intramuscular, enteral, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion of which intravenous (includingcontinuous intravenous administration) is most preferred) solutions inaqueous propylene glycol can be employed, as well as sterile aqueoussolutions of the corresponding water-soluble salts. Such aqueoussolutions may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. These aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal injection purposes. In this connection,the sterile aqueous media employed are all readily obtainable bystandard techniques well-known to those skilled in the art.

The products of the invention can also be administered intranasally orby inhalation and are conveniently delivered in the form of a dry powderinhaler or an aerosol spray presentation from a pressurised container,pump, spray, atomiser, nebuliser, with or without the use of a suitablepropellant.

Alternatively the products of the invention can be administered in theform of a suppository or pessary, or they may be applied topically inthe form of a gel, hydrogel, lotion, solution, cream, ointment orpowder. The products of the invention may be dermally or transdermallyadministered, for example, by use of a skin patch, depot or subcutaneousinjection. They may also be administered by pulmonary or rectal routes.

For oral administration, the pharmaceutical composition may be in theform of for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are capsules, tablets, powders, granules or asuspension, with conventional additives such as lactose; mannitol, cornstarch or potato starch; with binders such as crystalline cellulose,cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators such as corn starch, potato starch or sodiumcarboxymethylcellulose; and with lubricants such as talc or magnesiumstearate. The active ingredient may also be administered by injection asa composition wherein, for example, saline, dextrose or water may beused as a suitable carrier.

The products of the invention may also find application as/in an oralformulation wherein the product is formulated in a carrier, for exampleselected from films, tapes, gels, microspheres, lozenges, chewing gum,dentrifices and mouthwash.

The amount of therapeutically active compound that is administered andthe dosage regimen for treating a disease condition with the compoundsand/or compositions of this invention depends on a variety of factors,including the age, weight, sex and medical condition of the subject, theseverity of the disease, the route and frequency of administration, andthe particular compound employed, as well as the pharmacokineticproperties of the individual treated, and thus may vary widely. Thedosage will generally be lower if the compounds are administered locallyrather than systemically, and for prevention rather than for treatment.Such treatments may be administered as often as necessary and for theperiod of time judged necessary by the treating physician. One of skillin the art will appreciate that the dosage regime or therapeuticallyeffective amount of the inhibitor to be administrated may need to beoptimized for each individual. The pharmaceutical compositions maycontain active ingredient in the range of about 0.1 to 2000 mg,preferably in the range of about 0.5 to 500 mg and most preferablybetween about 1 and 200 mg. A daily dose of about 0.01 to 100 mg/kg bodyweight, preferably between about 0.1 and about 50 mg/kg body weight andmost preferably from about 1 to 20 mg/kg body weight, may beappropriate. The daily dose can be administered in one to four doses perday.

The products of the invention are preferably administered to therespiratory tract. Thus, the present invention also provides aerosolpharmaceutical formulations comprising a product of the invention. Alsoprovided is a nebuliser or inhaler containing a product of theinvention.

Additionally, the products of the invention may be suited to formulationas sustained release dosage forms and the like. The formulations can beso constituted that they release the active agents, for example, in aparticular part of the intestinal or respiratory tract, possibly over aperiod of time. Coatings, envelopes, and protective matrices may bemade, for example, from polymeric substances, such aspolylactide-glycolates, liposomes, microemulsions, microparticles,nanoparticles, or waxes. These coatings, envelopes, and protectivematrices are useful to coat indwelling devices, e.g. stents, catheters,peritoneal dialysis tubing, draining devices and the like.

The products of the invention may include synergistically effectiveamounts of each active agent defined herein. The invention thereforeincludes products comprising a synergistically effective amount of (i) afirst antibiofilm agent, (ii) a second antibiofilm agent which isdifferent from the first antibiofilm agent and is typically anantimicrobial peptide. The product may be for use in the manufacture ofa medicament, for simultaneous, separate or sequential administrationsaid agents in the treatment of a microbial infection for example abiofilm infection. “Synergistically”, as used herein, may describe theaction of the two or more agents of the product of the invention workingtogether to produce an effect greater than the expected combined effectof the agents used separately.

In a further aspect of the invention there is provided a substrate towhich a product of the invention is applied or attached. Preferably, thesubstrate is suitable for application to wounds or delivery to woundsites. Preferably, the substrate allows for the transfer of the activeagents of the product of the invention from the substrate to a wound bedto achieve their antibiofilm effect. The substrate may be a dressing,for example, wound dressing. The dressing may comprise a fabric materialor it may be a collagen-like material. The substrate may be in anysuitable form for application to a wound, typically the substrate may bein the form of a hydrogel, colloid, ointment, cream, gel, foam or spray.

The products of the invention may also find application as/in adisinfectant or biocide. In this context, the peptide or pharmaceuticalcompositions of the invention may be applied, either alone or incombination with other disinfecting agents, to a surface to be treated.As used herein a “surface to be treated” may be a substrate as definedherein and may include medical devices and indwelling devices, e.g.stents, catheters, peritoneal dialysis tubing, draining devices, jointprostheses, dental implants and the like.

Methods and Use

The invention provides a method of preventing biofilm formation in anenvironment comprising the step of administering to the environment aproduct according to the invention. The method may be in vivo or exvivo.

According to one embodiment, the method comprises the step ofadministering an antimicrobial peptide.

Advantageously the method comprises the step of administering a firstantibiofilm agent; and

a second antibiofilm agent different from the first one wherein at leastone of the first and second antibiofilm agents is an antimicrobialpeptide for example a cationic peptide.

The environment may comprise any biofilm forming microorganism selectedfrom bacteria, fungi, yeast, viruses and protozoa.

Typically the microorganism is a bacterium for example a Gram-positiveor Gram-negative bacterium. A bacterial pathogen may be derived from abacterial species selected from the group consisting of: Staphylococcusspp., e.g. Staphylococcus aureus, Staphylococcus epidermidis;Enterococcus spp., e.g. Enterococcus faecalis; Streptococcus pyogenes;Listeria spp.; Pseudomonas spp.; Mycobacterium spp., e.g. Mycobacteriumtuberculosis; Enterobacter spp.; Campylobacter spp.; Salmonella spp.;Streptococcus spp., e.g. Streptococcus Group A or B, Streptoccocuspneumoniae; Helicobacter spp., e.g. Helicobacter pylori; Neisseria spp.,e.g. Neisseria gonorrhea, Neisseria meningitidis; Borrelia burgdorferi;Shigella spp., e.g. Shigella flexneri; Escherichia coli; Haemophilusspp., e.g. Haemophilus influenzae; Chlamydia spp., e.g. Chlamydiatrachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Francisellafularensis; Bacillus spp., e.g. Bacillus anthraces; Clostridia spp.,e.g. Clostridium botulinum; Yersinia spp., e.g. Yersinia pestis;Treponema spp.; Burkholderia spp.; e.g. Burkholderia mallei and Bpseudomallei.

In particular the bacterium may include Pseudomonas spp., for examplePseudomonas aeruginosa; Staphylococcus spp., for example Staphylococcusaureus and Staphylococcus epidermidis; Haemophilus spp., for exampleHaemophilus influenza; Burkholderia spp., for example Burkholderiacepacia; Streptococcus spp., Propionibacterium spp., for examplePropionibacterium acnes. Preferably the bacterium is selected fromPseudomonas spp., for example Pseudomonas aeruginosa and Staphylococcusspp., for example Staphylococcus aureus and Staphylococcus epidermidis.

A viral pathogen may be derived from a virus selected from the groupconsisting of: Human Immunodeficiency Virus (HTV1 & 2); Human T CellLeukaemia Virus (HTLV 1 & 2); Ebola virus; human papilloma virus (e.g.HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 andHPV-56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus;Epstein Barr virus; influenza virus, hepatitis B and C viruses, Variolavirus, rotavirus or SARS coronavirus.

A parasitic pathogen may be derived from a parasitic pathogen selectedfrom the group consisting of Trypanosoma spp. (Trypanosoma cruzi,Trypanosoma brucei), Leishmania spp., Giardia spp., Trichomonas spp.,Entamoeba spp., Naegleria spp., Acanthamoeba spp., Schistosoma spp.,Plasmodium spp., Crytosporidiwn spp., Isospora spp., Balantidium spp.,Loa Loa, Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma ssp., e.gToxoplasma gondii. A fungal pathogen may be derived from a fungalpathogen which is of the genus Candida spp., (e.g. C. albicans),Epidermophyton spp., Exophiala spp., Microsporiim spp., Trichophytonspp., (e.g T. rubrum and T. interdigitale), Tinea spp., Aspergillusspp., Blastomyces spp., Blastoschizomyces spp., Coccidioides spp.,Cryptococcus spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrixspp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophoraspp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomaduraspp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveriaspp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp.,Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp.,Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp.,Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp.,Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucorspp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporiumspp., Scopulariopsis spp., Sporobolomyces spp., Syncephalastrum spp.,Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp.,Verticillium spp., Wangiella spp.

According to a further embodiment the microorganism may be a fungi, inparticular Candida.

The method of the invention may be used to minimise and, preferably,prevent the formation of biofilms in a variety of environmentsincluding, but not limited to, household, workplace, laboratory,industrial environment, aquatic environment (e.g. pipeline systems),medical devices including indwelling devices such as defined herein,dental devices or dental implants, animal body for example human body.

The method of the invention may thus be used in the mouth to prevent theformation of plaque or caries on a human tooth or dental implant forexample a denture.

The method of the invention may be used to prevent or restrict theformation of a biofilm in the human body especially in the treatment ofmicrobial infections. Conditions associated with biofilm infections mayinclude topical infections, oral infections and systemic infections.Topical infections may include wounds, ulcers and lesions for example,cutaneous wounds such cuts or burns, and conditions associatedtherewith.

Oral infections may include gingivitis, periodontitis and mucositis.

Systemic infections may include cystic fibrosis and other conditionsassociated with mucosal infections, for example, gastrointestinal,urogenital or respiratory infections.

Another aspect of the invention resides in methods of treating,preventing or delaying the progression of a disease or conditionassociated with the presence of a microbial biofilm infection in amammal, especially a human, by administering a therapeutically effectiveamount of a product of the invention to the mammal.

By an “effective” amount or “therapeutically effective amount” is meantan amount of one or more active substances which, within the scope ofsound medical judgment, is sufficient to provide a desired effectwithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

According to one aspect of the present invention the method comprisesthe step of administering an antimicrobial peptide.

Advantageously the method comprises the step of administering

a first antibiofilm agent; and

a second antibiofilm agent different from the first one wherein at leastone of the first and second antibiofilm agents is an antimicrobialpeptide for example a cationic peptide.

The invention further provides the use of a product of the invention inthe manufacture of a medicament for the treatment of a microbialinfection, particularly a microbial biofilm infection, by prophylaxis ortherapy of the combinations of active agents outlined above.

Additionally the present invention provides the use of the antimicrobialpeptide described above in the manufacture of a medicament for thetreatment of a microbial infection, particularly a microbial biofilminfection, by prophylaxis or therapy.

Thus the product of the invention may be useful in the prevention of,delay of progression of, or treatment of a disease or condition selectedfrom the group consisting of skin and wound infections, middle-earinfections, gastrointestinal tract infections, peritoneal membraneinfections, urogenital tract infections, oral soft tissue infections,formation of dental plaque, eye infections (including contact lensecontamination), endocarditis, infections in cystic fibrosis, andinfections of indwelling medical devices such as described herein.

The invention also includes methods of treatment in which a product ofthe invention is administered to a mammal together with one or moreother antibacterial agents for example an antibiotic.

The inventors have surprisingly found that certain dispersants, inparticular mucolytic agents, inhibit the growth of biofilm persistercells. Thus the invention also includes a method of treating/preventingbiofilm formation in an environment comprising administering to saidenvironment a mucolytic agent, for example cysteamine. The mucolyticagent may be administered alone or in combination with anotherantimicrobial agent for example an antimicrobial peptide.

The invention also provides a method for treating a microbial infection,particularly a microbial biofilm, by prophylaxis or therapy, comprisingthe administration in a therapeutically effective amount of adispersant, in particular a mucolytic agent, for example cysteamine.

The invention further provides the use of a dispersant, in particular amucolytic agent, for example cysteamine, in the manufacture of amedicament for the treatment of a microbial infection, particularly amicrobial biofilm infection.

The active agents mentioned in this specification can exist in differentforms, such as free acids, free bases, esters and other prodrugs, saltsand tautomers, for example, and the invention includes all variant formsof the agents.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to”, andare not intended to (and do not) exclude other moieties, additives,components, integers or steps.

Generally the term “approximately” is intended to encompass a range of10% or less of any numerical value to which it is applied.

Further aspects and embodiments of the invention are set forth in thefollowing description and claims.

EXAMPLES Activity of Antimicrobial Agents Against Bacterial Biofilms 1.Materials and Methods 1.1 Bacterial Strains

Pseudomonas aeruginosa ATCC27853, P. aeruginosa BAA-47 (PAO1), P.aeruginosa DSM1128, P. aeruginosa DSM1299 and S. epidermidis ATCC35984,S. epidermidis ATCC 12228 Staphylococcus aureus 25923 andmethicillin-resistant Staphylococcus aureus DSM 11729 (MRSA) (DSMZ,Braunschweig, Germany) were used in this study. Four P. aeruginosaclinical isolates (NH57388A-D, Hoffmann et al., 2005, 2007) wereobtained and used for antimicrobial susceptibility testing.

1.2 Preparation of Antimicrobial Compounds

The antimicrobial agents tested in this study were the cationic peptideNP108, which corresponds to a 10-20 kDa poly-L-lysine, hydrobromide andcysteamine (NM001). Both agents were obtained from Sigma-Aldrich(Gillingham, UK) and stock solutions were prepared at 20 mg/ml in 14-18MΩ·cm pure water (Purite HP40 water purification system, Oxon, UK). Oncedissolved, the preparations were filter-sterilized using 0.22 μm filters(Millipore, Watford, England) and stored at −20° C.

The following NovaBiotics antimicrobial peptides were also investigated

NP339  dRdRdRdRdRdRdRdRdRdRdRdRdR NP340 Ac-dRdRdRdRdRdRdRdRdRdRdRdRdR-CONH NP341 dRdRdRdRdRdRdRdRdRdRdRdRdR-CONH NP352  RRRRRRRRRRRRRRR NP432 RRRFRFFFRFRRR NP438  HHHFRFFFRFRRR NP441  HHPRRKPRRPKRRHH NP445 KKFPWRLRLRYGRR NP449  KKPRRKPRRPKRKK-cysteamine NP451 HHPRRKPRRPKRHH-cysteamine NP457  RRRRR-cysteamine NP458 RRRRRHH-cysteamine

NovaBiotics antimicrobial peptides were synthesized by NeoMPS(Strasbourg, France) using Fmoc synthesis and were at least 95% pure.

1.3 Preparation of the Bacterial Inoculum

The bacterial inoculum was established by the dilution method fromactively-growing cultures in Mueller-Hinton broth, standardized with 0.5McFarland turbidity standard as described in the CLSI method M26-A.

1.4 Determination of the Minimum Inhibitory Concentration (MIC)

To determine the prevention of biofilm formation, both the bacterialinoculum and the antimicrobial agents were added simultaneously to theplates. The plates were incubated at 37° C. for 24 h and the opticaldensity was read at 625 nm on a microtitre plate reader (BioTekPowerwave XS, Winooski, USA). The MIC was obtained as the lowestconcentration of antimicrobial showing total inhibition of bacterialgrowth.

1.5 Determination of the Fractional Inhibitory Concentration (FIC)

The FIC corresponds to an interaction coefficient indicating whether thecombination of antimicrobial agents is synergistic, additive, antagonistor neutral. The FIC is determined by comparing the activity of an agentin combination (MIC of agent A+agent B) with the activity of the agentalone (MIC of agent A or agent B) as follow (Singh et al., 2000):

FIC=MIC_(A[combination])/MIC_(A[alone])+MIC_(B[combination])/MIC_(B[alone])

Additive combinations of two antimicrobial agents are indicated by a FICindex of 1, whereas a FIC index<1 indicates synergistic combinations.Neutral combinations would give a FIC between 1 and 4, a FIC indexhigher than 4 indicates antagonist effects between the two antimicrobialagents.

The FIC was also calculated to assess the interaction of twoantimicrobial agents in combination against bacterial biofilms. The sameformulae applied, using MBEC instead of MIC.

1.6 Determination of the Minimum-Biofilm Eradication Concentration(MBEC)

A total volume of 100 μl bacterial inoculum in Mueller-Hinton was addedto each well of 96-well plates (challenge plates) and the plates wereincubated at 37° C. for 24 h on a gyrorotary shaking platform (Grant-bioPS-3D, Shepreth, England) at 24 rpm to allow for biofilm formation.

The challenge plates were then rinsed once with sterile PBS (lx) andtwo-fold serial dilutions of antimicrobial agents in Mueller-Hinton wereadded to the challenge plates. The challenge plates were incubated at37° C. for 24 h on a gyrorotary shaking platform (Grant-bio PS-3D,Shepreth, England) at 24 rpm.

The supernatants from each of the challenge plates were transferred intofresh plates and the optical density was measured at 625 nm on amicrotitre plate reader (BioTek Powerwave XS, Winooski, USA). The MBECwas obtained by the lowest concentration of antimicrobial showing nobacterial growth.

1.7 Estimation of the Persister Cells in the Biofilms

Following transfer of the supernatant from the challenge plates, thebiofilms were rinsed once with sterile PBS (lx) and 100 μl of BacLightlive/dead fluorescent staining solution (Invitrogen, Paisley, UK)containing 4 μM SYTO9 and 20 μM propidium iodide (PI) in sterile PBS(lx) were added to the wells of the challenge plates. The plates wereincubated at room temperature in the dark for 15 min and thefluorescence was read at 485(ex)/528(em) and 485(ex)/645(em) for SYTO9and PI fluorescence, respectively on a fluorescence microtitre platereader (BioTek Synergy HT, Winooski, USA) with the sensitivity set at 50and bottom optics position was selected. Direct observation of thebiofilms with an Axiovert 40 fluorescence microscope (Zeiss, Gottingen,Germany) allowed identifying the presence of live and dead bacteria andpictures of the biofilms were taken at 100 to 400-fold magnification.

The relative viability of persister cells was determined by thelive/dead fluorescence measurements ratio and microscopic observationswere used to confirm the presence or absence of live cells.

2. Results 2.1 Prevention of Biofilm Formation

In order to assess for the prevention of biofilm formation by bothGram-positive and Gram-negative bacteria, the bacterial inoculum andantimicrobial agents were added simultaneously in the plates. The rangeof concentrations of antimicrobial agents was 0-500 μg/ml NP108 and0-320 μg/ml cysteamine against the Gram-negative bacteria P. aeruginosaATCC BAA-47 and 0-1000 μg/ml NP108 and 0-320 μg/ml cysteamine againstthe Gram-positive MRSA.

2.1.1 Activity Against P. aeruginosa ATCC BAA-47

The MIC of NP108 was 62.5 μg/ml and 320 μg/ml for cysteamine. NP108 wasbactericidal at 250 μg/ml whereas cysteamine was not bactericidal at upto 320 μg/ml (data not shown).

In the presence of 160 μg/ml cysteamine the MIC of NP108 was reduced to31.25 μg/ml. When the concentration of cysteamine was doubled (ie. 320μg/ml) no growth was observed regardless of the concentration of NP108.

Determination of the FIC for this combination indicates that theantimicrobial agents have additive effects (FIC=1). Moreover,bactericidal activity was obtained in the presence of 125 μg/ml NP108and 320 μg/ml cysteamine (data not shown), which confirms the additiveeffect of these agents.

2.1.2 Activity Against S. aureus DSM 11729

The MIC of NP108 was 125 μg/ml and more than 320 μg/ml for cysteamine.NP108 was bactericidal at 125 μg/ml whereas cysteamine was notbactericidal at up to 320 μg/ml (data not shown).

Increasing concentrations of cysteamine showed a higher inhibition ofgrowth for any given concentration of NP108. In the presence of 40 μg/mlcysteamine the MIC of NP108 was reduced to 31.25 μg/ml and down to15.625 μg/ml when 320 μg/ml cysteamine were added.

Determination of the FIC for this combination indicates that theantimicrobial agents have at least additive effects (FIC<1). Moreover,bactericidal activity was obtained in the presence of 31.25 μg/ml NP108and ≧160 μg/ml cysteamine as well as 62.5 μg/ml NP108 and ≧80 μg/mlcysteamine (data not shown), which confirms the additive effect of theseagents.

Appendix 1 shows the time course activity of the short linear argininepeptides (NP339, NP340, NP341 and NP352) against S. aureus DSM 11729planktonic cells.

Appendix 2 provides a summary of the activity of NP108, cysteamine, bothcompounds in combination as well as the activity of NP339 and NP341against S. aureus DSM 11729 and P. aeruginosa BAA-47 planktonic cells.

2.2 Destruction of Formed Biofilms

The assessment of the activity of NP108 and cysteamine against bacterialbiofilms was carried out with 24 h-old biofilms and the activity of bothcompounds in combination was also determined. The activity of theantimicrobial agents against bacterial biofilms was determined by theiractivity against the biofilm cells and against the persister cells.

2.2.1 Activity of NP339 Against Bacterial Biofilms

FIG. 5 shows the high activity of NP339 against biofilms of 3Staphylococcus species, resulting in MBEC of 156 to 625 μg/ml. Theincrease in optical density at the highest dose of NP339 against S.aureus 25923 is likely to be an artefact due to the complex andheterogeneous nature of microbial biofilms. In contrast NP339 reducedthe growth P. aeruginosa BAA-47 (PAO1), but even the highest dose tested(i.e. 5 mg/ml) was not sufficient to inhibit 100% of the biofilm cells.

FIG. 6 provides evidence that NP339 is active against persister cells.In contrast to the activity of NP339 against the biofilm cells of the 4strains tested, it was less active against the persister cells ofStaphylococcus species than those of P. aeruginosa BAA-47 (PAO1). NP339was able to inhibit the viability of P. aeruginosa BAA-47 (PAO1)persister cells at 625 μg/ml.

2.2.2 Activity of NP341 Against Bacterial Biofilms

Similarly to NP339 (FIG. 5), NP341 showed significant reduction inbiofilm cells viability. The MBEC for MRSA 11729 and S. epidermidis12228 was 625 μg/ml. NP341 reduced the viability of biofilm cells ofMRSA 11729 and P. aeruginosa BAA-47 (PAO1) by a 2 to 3-fold factor.

As seen with NP339, the viability of P. aeruginosa persister cells wastotally inhibited at 625 μg/ml NP341. The viability of the persistercells of the 3 Staphylococcus species was decreased by 25 to 50%.

2.2.3 Activity of Cysteamine Against Bacterial Biofilms

FIG. 9 provides evidence that cysteamine has antimicrobial activityagainst biofilm cells of the Gram-positive and Gram-negative bacteriatested.

FIG. 10 shows the activity of cysteamine against persister cells of theGram-negative and Gram-positive bacteria tested.

The results presented here show the antimicrobial activity of the linearshort cationic peptides NP339 and NP341 against biofilms ofGram-positive and Gram-negative bacteria. These compounds appear moreeffective against the biofilm cells of Gram-positive bacteria thanGram-negative bacteria, whereas it is the opposite against persistercells. Cysteamine showed activity against biofilm cells at highconcentrations, however, it suppressed the viability of bothGram-positive and Gram-negative persister cells at the lowestconcentration tested (i.e. 6.25 mg/ml).

2.2.4 Activity of NP108 and Cysteamine in Combination Against P.aeruginosa ATCC BAA-47

The combination of these two antimicrobial agents showed completeinhibition of bacterial growth in the presence of 250 μg/ml NP108 and62.5 to 500 μg/ml cysteamine. The addition of 31.25 μg/ml cysteamine to500 μg/ml NP108 had a similar effect, whereas 31.25 μg/ml cysteamineplus 250 μg/ml NP108 showed only partial inhibition of bacterial growth.

The FIC obtained with those MBEC values (MBEC_(NP108[alone])>500 μg/ml,MBEC_(NP108[combination])=250 μg/ml, MBEC_(cysteamine[combination])=62.5μg/ml, MBEC_(cysteamine[alone])=>100,00 μg/ml), was ˜0.5, whichindicates a synergistic effect between these two antimicrobial agents.This is consistent with the observations made from the activity ofNP108/cysteamine combination against the planktonic cells (FIG. 2).

The activity of NP108 and cysteamine against the persister cells wasassessed using a fluorescence staining method to determine the relativeviability of the cells. The nucleic acid-binding fluorescent moleculesused were SYTO9 and PI, which penetrate all bacterial cells (greenfluorescence) and membrane-disrupted cells (red fluorescence),respectively. Therefore the ratio green (live)/red (dead) fluorescenceemitted gives an indication of the relative viability of the bacterialpopulation and is used to estimate the presence of residual live cellscorresponding to persister cells within the biofilm.

FIG. 12 shows that the relative viability of the biofilms treated witheither NP108 or cysteamine remained significant, indicating the lack ofactivity of these compounds against the persister cells of P. aeruginosaATCC BAA-47.

FIG. 13 provides evidence that the combination of NP108 and cysteamineshowed higher activity against the persister cells of P. aeruginosa ATCCBAA-47 than either compound alone (FIG. 12). The most efficientcombinations against those cells were 250-500 μg/ml NP108 and 62.5-500μg/ml cysteamine. These combinations showed the lowest relativeviability within the biofilms. Similar results were obtained with 31.25μg/ml NP108 and 500 μg/ml cysteamine with only partial inhibitionobserved with 250 μg/ml cysteamine.

The activity of these compounds against persister cells showssimilarities to the profile of optimum combinations obtained against thebiofilm cells (FIG. 11). Moreover, direct microscopic observations ofthe fluorescently-stained biofilms confirmed the activity of thesecombinations against the persister cells as no live cells could beobserved in the presence of 250-500 μg/ml NP108 and 62.5-500 μg/mlcysteamine (data not shown).

2.2.5 Activity of NP339 and Cysteamine in Combination Against P.aeruginosa

FIG. 14(a)-(d) show the activity of 3 concentrations of NP339: 1 μg/ml,10 μg/ml and 100 μg/ml in combination with increasing concentrations ofcysteamine up to 10 mg/ml against 4 strains of Pseudomonas aeruginosa.

These data clearly demonstrate the increased antimicrobial activityagainst P. aeruginosa biofilm cells of NP339 in combination withcysteamine. The following figures show examples of the activity of thesecombinations against persister cells of 2 of these strains.

FIG. 16 shows the activity of NP108 and cysteamine against S. aureus DSM11729 biofilm cells. The MBEC for cysteamine was 250 μg/ml, whereasNP108 inhibited the growth of those cells at 125 μg/ml.

The combination of NP108 and cysteamine showed complete inhibition ofbacterial growth in the presence of 31.25 μg/ml NP108 and 62.5 μg/mlcysteamine and partial inhibition with lower concentrations of eithercompound (FIG. 17). Hence, the FIC obtained with those MBEC(MBEC_(NP108[alone]) 125 μg/ml, MBEC_(NP108[combination])=31.25 μg/ml,MBEC_(cysteamine[alone])=250 μg/ml, MBEC_(cysteamine[combination])=62.5μg/ml) was 0.5 thereby indicating a synergistic effect between these twoantimicrobial agents against biofilm of these Gram-positive bacteria.Similar results were observed for Gram-negative bacterial biofilm (FIG.11). This is also consistent with the observations made from theactivity of NP108/cysteamine combination against the planktonic cells ofS. aureus DSM 11729 (FIG. 4).

Similarly to the lack of activity observed against P. aeruginosa ATCCBAA-47 persister cells (FIG. 12), the relative viability of the S.aureus DSM 11729 biofilms treated with either NP108 or cysteamineremained significant, indicating the lack of activity of these compoundsat low concentrations against the persister cells of these Gram-positivebacteria (FIG. 18).

The combination of NP108 and cysteamine showed higher activity againstthe persister cells of S. aureus DSM 11729 than either compound alone(FIG. 19). The most efficient combinations against those cells were250-500 μg/ml NP108 and 125-250 μg/ml cysteamine. These combinationsshowed the lowest relative viability within the biofilms. Similarresults were obtained with 62.5 μg/ml NP108 and 500 μg/ml cysteamine.Combinations with lower concentrations of either compound showed highrelative viability within the biofilms.

Unlike the Gram-negative persister cells, direct microscopicobservations of the fluorescently-stained S. aureus DSM 11729 biofilmsindicated the presence of residual live cells at the highest combinedconcentrations of NP108 and cysteamine (data not shown).

Table 1 provides a summary of the activity of the short argininepeptides NP339, NP341, the poly-L-lysine NP108, cysteamine and thecombination of NP108 with cysteamine against a Gram-positive andGram-negative bacteria.

TABLE 1 Table 1: Summary of the activity of the tested antimicrobialagents against the Gram-negative P. aeruginosa strains and theGram-positive Staphylococcus spp. The number into brackets indicates themaximum number of strains tested. MIC: minimum inhibition concentration;MBEC: minimum biofilm eradication concentration; FIC: fractionalinhibitory concentration. P. aeruginosa strains Staphylococcus spp (7)(4) FIC: MIC (μg/ml) NP108 31.25-500    16-125 NP339 62.5  4-128 NP34131.25 250 Cysteamine  300-2,500 300-625 NP108/ 31.25/160 31.25/40Cysteamine NP108/ 1 0.6 Cysteamine FIC: MBEC (μg/ml) NP108 250->500125-250 NP339 >5,000 156-625 NP341 >5,000   625->5,000Cysteamine >5,000 >25,000 NP108/ 125/125-250/62.5  31.25/62.5-125/125   Cysteamine NP108/ ≦0.75 0.5-1   Cysteamine FIC: Persisters (μg/ml) NP108250->500 >500 NP339 625   625->5,000 NP341 625   625->5,000 Cysteamine 500-6,250  6,250-12,500 NP108/ 62.5/250-250/62.5  >250/>250 CysteamineNP108/ 0.75-1    ≦0.5 Cysteamine Notes: appendix 1 shows the MIC of thetested short arginine antimicrobials against Staphylococcus aureus DSM11729. appendix 2 shows the activity of the mucolytic agents cysteamineand N-acetylcysteine in combination with NP341 against P. aeruginosaATCC27853.

APPENDIX 1

The data (not shown) demonstrates the activity of short linear argininepeptides over a 48-h period against planktonic cells ofmethicillin-resistant S. aureus (MRSA) DSM 11729. The range ofconcentrations tested as shown in the legends is in mg/ml. The data (notshown) demonstrates the activity of short linear arginine peptides overa 48-h period against planktonic cells of methicillin-resistant S.aureus (MRSA) DSM 11729. The time course activity demonstrates thatbacterial growth inhibition is associated with the dose of antimicrobialand to the time of exposure to the cells. Complete bactericidal activitywas observed for NP339, NP 340 and NP352 at concentrations above 0.5mg/ml for the 48-h period; 0.125 and 0.25 mg/ml showed completeinhibition for at least 24 h, and lower concentrations such as 0.06 and0.03 mg/ml showed complete inhibition for at least 20 h and 15 h,respectively. Similar results were obtained with NP341, except that 0.25mg/ml showed complete inhibition for the 48-h period.

APPENDIX 2

In combination with 3-6 mg/ml of N-acetylcysteine, however, only 205μg/ml of NP341 are needed to reach the MBEC (FIG. 20a ). Similarincreased activity for the combination of these 2 compounds was observedagainst persister cells: 1024 μg/ml NP341+3128 μg/ml N-acetylcysteineinhibited approximately 75% of the persister cells, which is a muchhigher inhibition than that obtained with either of the two compoundsalone (FIG. 20b ).

The combination of cysteamine or N-acetylcysteine with NP341 showsincreased antibacterial activity compared to the activity of eithercompound alone. The MBEC of NP341 alone against P. aeruginosa ATCC27853was more than 2 mg/ml and more than 100 mg/ml for cysteamine (FIG. 21a). This indicates that there is no cooperative effect between the twocompounds against the biofilm cells of P. aeruginosa ATCC27853. However,such cooperation was observed against the persister cells: 205 μg/mlNP341+3 mg/ml cysteamine inhibit approximately 75% of the persistercells, which is much higher than any of the two compounds alone (FIG.21b ).

When used in combination with NP339, we observed that the addition ofcysteamine even in small amounts helps reducing the MBEC values of NP339(FIG. 14a-d ). More interestingly, the combination of NP339 andcysteamine also showed increased activity against persister cells of P.aeruginosa DSM1128 and P. aeruginosa BAA-47 (FIG. 15a-b ).

1.-51. (canceled)
 52. A method of treating a biofilm in a patient, byprophylaxis or therapy, comprising administering to the patient amucolytic agent wherein the mucolytic agent is cysteamine.
 53. Themethod of claim 52 wherein the patient has cystic fibrosis.
 54. Themethod of claim 52 wherein the biofilm is associated with an infectionin cystic fibrosis.
 55. A method of treating a condition associated withcystic fibrosis, by prophylaxis or therapy, in a patient comprisingadministering to said patient a mucolytic agent wherein the mucolyticagent is cysteamine.
 56. The method of claim 55 wherein the mucolyticagent is administered alone.
 57. A method of treating a bacterialinfection in a cystic fibrosis patient comprising administering to saidpatient an amount of cysteamine effective to treat said infection.